Nanoplasmonic imaging technique for the spatio-temporal mapping of single cell secretions in real time

ABSTRACT

A label-free method for the spatio-temporal mapping of protein secretions from individual cells in real time by using a chip for localized surface plasmon resonance (LSPR) imaging. The chip is a glass coverslip compatible for use in a standard microscope having at least one array of functionalized plasmonic nanostructures patterned onto it. After placing a cell on the chip, the secretions from the cell are spatially and temporally mapped using LSPR imaging. Transmitted light imaging and/or fluorescence imaging may be done simultaneously with the LSPR imaging.

PRIORITY CLAIM

The present application is a continuing application of U.S. applicationSer. No. 14/207,927, filed on Mar. 13, 2014 by Marc P. Raphael et al.,entitled “Nanoplasmonic Imaging Technique for the Spatio-TemporalMapping of Single Cell Secretions in Real Time,” which claimed thebenefit of U.S. Provisional Application No. 61/778,652, filed on Mar.13, 2013 by Marc P. Raphael et al., entitled “Nanoplasmonic ImagingTechnique for the Spatio-Temporal Mapping of Single Cell Secretions inReal Time” and U.S. Provisional Application No. 61/839,428, filed onJun. 26, 2013 by Marc P. Raphael et al., entitled “Silicon Backing Ringand Multiplexing Applications for LSPR Imaging.” The entire contents ofall of these applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to paracrine signal measuringand, more specifically, to label-free mapping of single cell secretionsin real time.

Description of the Prior Art

Paracrine signaling is a form of close-range communication betweencells, typically mediated by the secretion of proteins. The types ofproteins secreted as well as their spatial and temporal distributionsgive rise to a broad range of possible responses amongst the receivingcells, including cell migration and proliferation. Not surprisinglythen, paracrine signaling is found to play a central role in a diverserange of processes such as wound healing, angiogenesis and immuneresponse, which rely heavily on cell movement and division. The abilityto map the spatio-temporal nature of individual cell secretions is thusfoundational to understanding these processes.

There are, however, a number of roadblocks encountered in trying tomeasure paracrine signaling due to the proteins being both highlylocalized and external to the cell. While fluorescent fusion proteintags are now standard for tracking intracellular signaling, the approachis problematic for studying secreted proteins. First, the presence of arelatively large tag (27 kDa for GFP) may hamper the cell's ability tosecrete the protein of interest. Second, even if the molecule and itsfluorescent protein tag are successfully secreted, the result is adiffuse glow in the vicinity of the cell that is difficult to trackquantitatively in space and time.

As a result, direct measurements of secreted proteins from individualcells are typically performed using techniques founded uponimmunosandwich assays that either use fluorescent antibodies orcolormetric enzymatic reactions. While in the past such measurements hadtime resolutions on the order of days, technological advances thatcouple immunosandwich assays with lithographically patterned microwellsand microfluidics have enabled quantitative secretion monitoring withtime resolutions on the order of hours. Such advances have exposedcyclical behaviors in the rates at which stimulated T cells secretecytokines, and in a more general sense, have demonstrated how improvingtime resolutions can enhance the understanding of intercellularsignaling. Improved temporal resolutions hold the promise of detectingthe time for individual cells to begin secretion after externalstimulation, correlating secretion rates with stages of the cell cycleand, as we show in the present invention, distinguishing burst-likesecretions from those that are more steady state in nature.Immunosandwich-based assays are now capable of measuring hundreds orthousands of individual cells per experiment but their temporalresolution is limited by the introduction of the antibody probe whichnecessarily halts or ends the secretion study. A complimentary techniquethat focuses on a small number of cells but with higher spatial andtemporal resolution promises to help complete the picture of close rangecell-to-cell communication by bridging the time scale gap from secondsto days.

BRIEF SUMMARY OF THE INVENTION

The aforementioned problems are overcome in the present invention whichprovides a label-free method for the spatio-temporal mapping of proteinsecretions from individual cells in real time by using a chip forlocalized surface plasmon resonance (LSPR) imaging. The chip is a glasscoverslip compatible for use in a standard microscope having at leastone array of functionalized plasmonic nanostructures patterned onto it.After placing a cell on the chip, the secretions from the cell arespatially and temporally mapped using LSPR imaging. Transmitted lightimaging and/or fluorescence imaging may be done simultaneously with theLSPR imaging.

The application of nanoplasmonic imaging to the study of extracellularsignaling brings with it a number of advantages: (1) The proteinsecretions are measured in real-time with the frequency of time pointslimited only by the exposure time of the camera, typically 250-400 ms.(2) The Au plasmonic nanostructures are lithographically patterned ontostandard glass coverslips enabling more traditional imaging techniquessuch as fluorescence and bright field imagery to be readily integratedinto the experiments. Thus, morphological changes and intracellularfluorescent tags can be monitored simultaneously in real time. (3) Thenanostructures are calibrated for the quantitative determination ofsecreted protein concentration as a function of time and space. (4)Arrays of Au nanostructures positioned sufficiently far away from thecells can be utilized as control arrays used to distinguish globalvariations in signal from localized cell secretions. (5) The techniqueis applicable to both adherent and non-adherent cell lines. (6) Unlikefluorescent probes. Au plasmonic nanostructures do not exhibit blinkingor photobleaching, both of which are problematic in the previous methodsdescribed above.

There is currently no alternative method for the label-free and realtime imaging of protein secretions from individual cells that integratesthe standard glass coverslips widely used for cell imaging and culture.The label-free technique described herein enables secretion studies withtime resolutions on the order of hundreds of milliseconds and withoutlabeling, whereas the commercially available sandwich assay-basedtechniques have time resolutions on the order of hours or days andrequire fluorescent tags or enzyme-based colormetric probes.

These and other features and advantages of the invention, as well as theinvention itself, will become better understood by reference to thefollowing detailed description, appended claims, and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show principles of localized surface plasmon resonance(LSPR) imaging and the single cell secretion measurement. FIG. 1A showsthe nanoplasmonic response of a c-myc functionalized array to 200 nM ofcommercial anti-c-myc in serum-free media, introduced microfluidically.The mean intensity of the array was calculated within a 9.5×9.5 μmregion-of-interest centered about the array for each time point. Theillustrations highlight two response regimes for the functionalizednanostructures, which are depicted as cylinders. In the lower regime,only a small fraction of available c-myc peptides (spheres) are boundwith anti-c-myc (low fractional occupancy, f) while almost all areoccupied in the upper regime (f→1). No cells were present in thisexperiment. FIG. 1B is an illustration of an antibody-secreting cell inregistry with two nanoplasmonic arrays. The chip is loaded onto aninverted microscope and the fact that the majority of the substrate istransparent glass allows for live cell imaging using transmitted lightand fluorescence microscopy in parallel with the LSPR-based imagingtechnique used to measure the secretions.

FIGS. 2A-2D show secretion burst from a single cell. FIG. 2A is anoverlay of LSPR and TL images with the cell visible next to Array A as aresult of the TL illumination while the nanoplasmonic arrays areilluminated in LSPR mode. FIG. 2B shows normalized LSPR image intensity(Î_(cell)) of Arrays A-D. The distances from the center of the cell tothe center of each array were 15.4 μm, 39.2 μm, 72.2 μm, and 106 μm forArrays A, B, C and D, respectively. The Î_(cell) values have been offsetto be equal before the burst (t≦7800 s) so that the detection time andintensity of the burst at each array can be more readily compared. FIG.2C shows the mean intensity of Array D, highlighting the end of theexperiment at which 250 nM of commercial anti-c-myc was introduced forthe purpose of normalizing the response of the arrays. FIG. 2D shows anoverlay of LSPR and fluorescence images exposing portions of the cellmembrane labeled with Lissamin Rhodamine B. Scale bar: 10 μm.

FIGS. 3A-3C show a single cell secretion study. FIG. 3A is an overlay oftransmitted light and LSPR images highlighting the location of the cellrelative to 12 arrays. FIG. 3B shows the normalized LSPR responses ofArrays A, B and C (Î_(cell)) minus the average normalized response ofthe three control Arrays D, E and F (Î_(control)). The centers of ArraysA, B and C were located 11 μm, 23 μm and 35 μm, respectively, from thecenter of the cell. Arrays D, E and F were all located at least 65 μmfrom the center of the cell. FIG. 3C shows an overlay of the two images(LSPR, transmitted light) with a spatial map of secreted antibodyconcentrations as generated by finite element analysis. For thecalculation, the simulated cell was 16 μm in diameter, had an adhesionspot of 5.5 μm and secreted antibodies uniformly at 1000 antibodies/s.The concentration scale has units of pM and the distance scale bar is 10μm.

FIG. 4 shows a comparison of the time-dependent secretions from foursingle cell studies, all of which were within 15 μm of an array. Thenormalized LSPR image intensity of the array (Î_(cell)) minus thenormalized intensity of a control array (Î_(control)) is plotted versustime. FIG. 4 also shows the fractional occupancy, f, of the arrayclosest to Cell A as determined from the LSPR spectra which wascollected simultaneously with the LSPR imagery. A concentration of312±89 pM was calculated using the data in the period from 100 min to135 min (highlighted region) at which f was constant with time.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a label-free approach based uponlocalized surface plasmon resonance (LSPR) imaging for the real-timemeasurement of protein secretions from individual cells. LSPR biosensingis founded upon the fact that the plasmonic resonance of a metallicnanostructure exhibits both a red shift and an increase in scatteringintensity when analyte binding creates small perturbations in the localindex of refraction. When imaged on a CCD camera these spectroscopicsignatures are manifested as an increase in the brightness of thenanostructures (FIG. 1A) and can be quantified in terms of thefractional occupancy of surface bound receptors. In contrast tothin-film based surface plasmon resonance (SPR) approaches that requiretotal internally reflected light for the excitation of the surfaceplasmons, nanoplasmonic resonances can be excited with visible lightusing the same optical configurations used in traditional wide-fieldmicroscopy setups (FIG. 1B).

The approach of the present invention has been validated by using LSPRimaging to spatially and temporally map the secretion of anti-c-mycantibodies from individual 9E10 hybridoma cells. Square arrays of goldnanostructures were patterned onto No. 1.5 glass coverslips usingelectron-beam nanolithography as described in Raphael et al., “A NewMethodology for Quantitative LSPR Biosensing and Imaging,” Anal. Chem.,84, 1367-73 (2012), the entire contents of which is incorporated hereinby reference. Each array (6× 6 μm) consisted of 400 evenly spacednanostructures separated by a pitch of 300 nm. The bases of thenanostructures were circular in cross section with diameters of 70±5 nmand the heights were 75±2 nm, which gave a plasmonic resonance peakcentered about 625 nm when immersed in serum free cell culture media(SFM). The arrays were separated by 33 μm allowing for as many as 12arrays to be incorporated into the field-of-view (FOV) when using a 63×microscope objective and as many as 35 arrays in the FOV when using a40× objective. As such, 97% of the FOV was transparent glass, allowingfor the cells on the glass portion to be viewed by traditionalmicroscopy techniques such as fluorescence and transmitted light (TL)imaging. The remaining 3% was patterned with the Au nanostructure arraysand utilized for cell secretion measurements by LSPR imaging. Inaddition to the imagery, a beam splitter was placed before the CCDcamera directing half the collected light to a fiber-optically coupledspectrometer. The fiber was aligned to collect spectra from the arrayclosest to the cell and this spectral information was analyzed todetermine the fractional occupancy of surface bound receptors. One dataset was collected per minute comprising a combination of three images(LSPR, TL, and fluorescence) as well as the LSPR spectrum of theindividual array.

The patterned coverslips were cleaned by hydrogen plasma ashing, and thegold nanostructures were functionalized with a two-componentself-assembled monolayer (SAM) of thiols consisting of a 3:1 ratio ofSH—(CH₂)₈-EG₃-OH (SPO) to SH—(CH₂)₁₁-EG₃-NH₂ (SPN). The SPN componentwas covalently conjugated with commercially available c-myc peptide. Thefunctionalized chips were mounted onto a custom built microfluidicperfusion assembly for the introduction of fresh media and loaded onto aZeiss Axio Observer inverted microscope. The assembly was enclosedwithin an incubation chamber regulated with 5% CO₂ and 98% humidity at37° C.

In LSPR imaging mode, the arrays appear as bright squares on a darkbackground and only the portion of the cell adherent to the surface isvisible, while in TL imaging the whole cell is visible and can bemonitored for morphological changes (FIG. 2A). The one minute timeresolution enabled the detection of bursts of secreted antibodies inLSPR imaging mode that otherwise would have gone undetected (FIG. 2B).In particular, at the 130 min mark of the experiment, the cell that hadgrown steadily to a diameter of 27 μm, contracted to 25 μm within the 1minute time span between data points. Simultaneously, a sharp increasein the LSPR imaging signal was detected at the closest array (FIG. 2B,Array A). Arrays B and C also detected a pulse, time delayed by 1 minand 3 min, respectively. The fact that the size and the slope of thesignal decreased with increasing distance between cell and array isconsistent with a pulsed wave of antibodies originating at the cell anddiffusing outward. The diffusing wave was readily measured at arrayslocated at distances greater than 70 μm from the cell.

To account for variations in array intensity and dynamic range, theresponse of each array was individually normalized. The procedureconsisted of introducing a saturating solution of commercial anti-c-mycantibodies (250 nM) at the end of the experiment (FIG. 2C). The meanintensity, I(t), within a 9.5× 9.5 μm region-of-interest (ROI) centeredabout the array was then normalized at each time point, t, with theequation Î(t)=[I(t)−I(t_(o))]/[I(t_(f))−I(t_(o))], where I(t_(o)) andI(t_(f)) were the mean ROI intensities at the beginning of theexperiment and after saturation, respectively.

Having both spatial and temporal information for the traveling waveformenabled the diffusion constant for the secreted antibodies to beestimated. Assuming a spherical emitter producing an outwardlypropagating pulse of antibodies with a Gaussian concentration profile,the onset of the measured pulse was associated with the peak of thewave. In this limit, D=r²/6·t, where D is the diffusion constant, r isthe distance from the center of the cell to the center of the array andt is the elapsed time from when the cell contracted. Analyzing allarrays in the FOV that detected the pulse, a range was obtained for D of0.6×10⁻⁷ cm²/s<D<5.5×10⁻⁷ cm²/s, which is consistent with the value of4×10⁻⁷ cm²/s measured for IgG antibodies in buffered saline solution.The calculated range in D is in large part a result of the uncertaintyin t, due to the 1 minute time resolution chosen for this experiment, aswell as the association of the onset of the signal at the array with aparticular feature of the Gaussian wave front. Nevertheless, thedetection of a traveling wave of secreted proteins from a single celland the ability to estimate D is illustrative of the generalapplicability of the present invention for the spatio-temporal mappingof paracrine signaling.

The cell plasma membrane was also labeled with the membrane-localizingdye Lissamin Rhodamine B, allowing for fluorescence-based imaging ofmembrane dynamics to be co-monitored with the LSPR and TL imaging (FIG.2D). The ability to integrate fluorescence microscopy with LSPR imagingserves is an example of how the present invention enableswell-established fluorescence methods for intracellular studies to beintegrated with extra-cellular secretion investigations.

The majority of cells studied exhibited continuous secretions over thecourse of the experiment as opposed to bursts (FIGS. 3A-3C). Thesecretions from individual cells were isolated and quantified witharrays adjacent to the cell by normalizing the response of each array inthe FOV as described above and then using the arrays located furthestfrom the cell as controls for background subtraction (FIG. 3B). Antibodysecretions were observed within minutes of the start of the experimentand, as expected, the signal strength diminished with increasingdistance between cell and array. In comparison to the secretive burststhat were detectable at distances greater than 70 μm from the cell,continuous secretions were detected at distances of 40 μm or less.Additional stochastic noise was often observed at the array closest tothe cell (FIG. 3B, Array A) due to changes in the cell's morphology thatcreated variations in the scattered light at the edge of the cell. Inthe event that the cell spread over the surface during the experimentand made contact with the array, the scattered intensity from theplasmonic nanostructures increased measurably at the edge of the arrayadjacent to the cell, in marked contrast to the detection of secretedantibodies in which the array intensity increased uniformly.

The data analysis procedure for background subtraction was important forisolating continuous-type secretions from individual cells. The purposeof the background subtraction was to eliminate global changes in thesignal that affect all arrays in the FOV over the course of theexperiment, such as volumetric changes in the media composition, focusdrift and variations in light source intensity. Arrays sufficientlydistant from the cell were insensitive to its secretions and thus couldserve as control arrays, conveniently integrated into the sameexperiment by the lithographic process. To help determine the minimumrequired separation distance between the cell and the control arrays,finite element analysis (FEA) was used to solve the diffusion equationin the vicinity of a model cell emitting antibodies at a constant rate.The secretion rate of 1000 antibodies/s used in the calculations was anexperimentally-determined average based on bulk secretion rate studiesof 4×10⁶ cells. The FEA results (FIG. 3C) show that a separation betweenthe cell and an array of greater than 65 μm reduces the secretedantibody concentration below the detection limit for the time scalesunder investigation (˜100 pM). This agreed with the experimentalobservation that Arrays D, E, and F, located 70 μm, 68 μm, and 81 μmfrom the center of the cell, respectively, had normalized responses thatwere statistically indistinguishable over the course of the three hourexperiment.

From a collection of single cell measurements of this type, localconcentrations were measured that varied from as high as a 312±89 pMabout some cells to below the detection limit of the array for others(FIG. 4). In each study, the cell was adjacent to the array with thecenter of the cell less than 15 μm from the array. The concentration atthe array was calculated using c(f)=K_(D)f/(1−f) where f is thefractional occupancy of surface-bound c-myc on the array determined fromthe simultaneously collected spectroscopic data of the LSPR peakresponse and K_(D) is the equilibrium dissociation constant of 1.8 nM.The equation is valid if f is constant with time, a condition that wasoften applicable two to three hours into the measurement (FIG. 4) andwhich is in agreement with FEA predictions that the concentration ofantibodies surrounding a continuously secreting hybridoma cell closelyapproaches a steady state within 30 min of the cell being introduced toa new environment.

The experimental technique described herein enabled the quantitativespatio-temporal mapping of secreted proteins from one to three cells perexperiment. As such, it stands as a complementary approach tohigh-throughput, single-cell immunosandwich assay techniques thatmeasure hundreds or thousands of individual cells but with lowerspatio-temporal resolutions. In addition, the chip architecture isdesigned to mimic that of a glass-bottomed culture dish setup. As such,polymer matrices (i.e. fibronectin, collagen) can be added to thesubstrate to enable adherent cell studies. We expect an amplified signalfrom cells resting on such a matrix and located directly over an arraydue to the trapping of the secreted proteins between the cell membraneand the substrate, thus allowing for the measurement of lower secretionrates. Many immunosandwich assays also incorporate multiplexing for thesimultaneous detection of multiple analytes, which may be done by spotprinting specific antigens to designated arrays. Finally, the label-freenature of LSPR imaging as well as its compatibility with TL andfluorescence imaging techniques gives experimental flexibility in thateither modified or unmodified cells can be investigated.

Methods Electron Beam Lithography of Au Nanostructures

The e-beam resists used for lithography were polymethyl methacrylate 4%in anisole (PMMA A4) and 6% ethyl lactate methyl methacrylate copolymer(MMA EL6), both from Microchem. The chromium etchant CR-7 was purchasedfrom Cyantek and methyl isobutyl ketone (MIBK)+isopropyl alcohol (IPA)in a 1:2 ratio was used for developing the resists. The substrates usedfor patterning the nanostructures were 25 mm diameter glass coverslipswith a nominal thickness of 170 μm. The cleaning of the coverslips,deposition of a chromium thin film to prevent charging and spinning ofthe resist bilayer have all been previously described in Raphael et al.,“A New Methodology for Quantitative LSPR Biosensing and Imaging,” Anal.Chem., 84, 1367-73 (2012). Samples were patterned via electron beamlithography using area doses in the range of 200 to 400 μC/cm². Thesamples were developed for 1 minute in an IPA/MIBK bath and the chromiumlayer wet-etched from the bottom of the pattern using the CR-7 etchant.A Ti/Au layer was deposited using a Temescal electron-beam evaporator.Following the metal deposition or etching, the PMMA/copolymer bilayerwas removed by soaking in acetone for 4 hours.

Au Nanostructure Cleaning and Functionalization

The chip was cleaned by plasma ashing at 40 W in 300 mTorr of a 5%hydrogen, 95% argon mixture and then functionalized by immersion in atwo-component ethanolic-based thiol solution (0.5 mM), consisting of a3:1 ratio of SH—(CH₂)₈-EG₃-OH to SH—(CH₂)₁₁-EG₃-NH₂ for 18 hours(Prochimia). The SPN component of the SAM layer was first reacted with a10 mg/mL solution of the heterobifunctional crosslinkersulfo-N-succinimidyl-4-formylbenzamide (Solulink) in PBS buffer (pH 7.4)and then conjugated to the c-myc peptide (HyNic-c-myc-tag, Solulink) inPBS buffer (pH 6.0) according to the manufacturer's instructions.Commercially obtained anti-c-myc antibodies (Genscript) were used forsaturating the surface bound c-myc and normalizing array response at theend of each experiment.

LSPR, Transmitted Light, and Fluorescence Microscopy

All imagery was acquired using Zeiss AxioVision software, an invertedZeiss Axio Observer microscope, and a thermoelectrically-cooled 16 bitCCD camera with 6.45×6.45 μm sized pixels (Hamamatsu ORCA R²).Experiments utilized either a 63× oil immersion objective (FIGS. 1A-B,2A-D, and 3A-C) or a 40× oil immersion objective (FIG. 4) and Koehlerillumination. The camera was operated in 2×2 binning mode, giving imageresolutions of 323 nm and 205 nm for the 40× and 63× objectives,respectively. CCD-based LSPR imaging and LSPR spectra were collected ina reflected light geometry using a 100 W halogen lamp for illuminationand crossed-polarizers to reduce the background contribution fromsubstrate-scattered light. Imagery and spectra were obtainedsimultaneously by placing a beam splitter at the output port of themicroscope and a long-pass filter with a 593 nm cut-off wavelength wasplaced before the CCD camera. For the spectral measurements, the focusedimage of the entire nanostructure array was projected on to the end of a600 μm diameter optical fiber and the spectra were subsequently measuredwith a spectrophotometer (Ocean Optics QE65000). Transmitted lightillumination was obtained with the same configuration but using a 100 Whalogen light source located above the chip. Fluorescence imagery wasacquired using a 540-580 nm LED module (Zeiss Colibri) and a filter cubeoptimized for rhodamine fluorescence. Exposure times for LSPR imaging,transmitted light imaging, fluorescence imaging and spectra collectionwere 300 ms, 300 ms, 1 sec and 1 sec, respectively. TL images werecontrast enhanced and false color was added to the grey-scalefluorescence images to better visualize the cell. All light sources wereshuttered when data was not being acquired to minimize the possibilityof phototoxic effects on the cells.

Microscope Incubation Environment and Stability

The microscope was equipped with a temperature controlled enclosure thatkept the stage temperature at 37.0±0.04° C. (Zeiss). An additionalincubation enclosure over the sample regulated the humidity and CO₂content to 98% and 5%, respectively. Under these conditions, the driftsin the x, y and z directions were less than 3 nm/min. Focus drift waslargely, though not entirely, corrected for during the experiment usinga Zeiss Definite Focus System. In plane drift was corrected withcommercially available post-experiment image alignment software (ZeissAxio Vision).

Hybridoma Cell Culturing and Labeling

Hybridoma cells (clone 9E10, ATCC) were cultured in complete growthmedium (RPMI-1640, ATCC) supplemented with 10% fetal bovine serum and 1%antibiotic/antimycotic (Sigma) using T75 flasks in a humidified tissueculture incubator at 37° C. under 5% CO₂ atmosphere. Cells weremaintained at a density of 3-5×10⁵ cells/mL and a subculture wasperformed every two days to maintain cell viability at 90-95%. Celldensities and viability were determined using a Countess automated cellcounter (Invitrogen). Before being introduced on to the microscope, thecells were harvested in complete growth medium, counted and theviability was assessed. All cell preparations used for imaging had >92%viability. Cells were pelleted by centrifugation (3000 rpm×5 min),washed twice with RPMI-1640 SFM to remove secreted antibodies andadjusted to a cell density of 2×10⁶ cells/mL. For imaging, 75 μL of cellsolution was manually injected into the imaging chamber. After 5minutes, typically 50-75 cells had adhered to the surface; the remainingcells were washed away with fresh SFM using the microfluidic perfusionsetup. For fluorescence imaging, the plasma membrane of live cells waslabeled with the membrane-localizing dye Lissamin rhodamine B1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammoniumsalt (rhodamine DHPE) (Ex, 560 nm/Em, 580 nm) (L-RB, Invitrogen). Thecells were washed and incubated for 20 min with 10 μM L-RB in Dulbecco'Modified Eagles Medium containing 25 mM HEPES (Invitrogen) on a rotatingshaker at room temperature. Following incubation, cells were washedtwice with SFM and prepared for imaging as described above.

Finite Element Analysis

Finite element analysis was conducted using FlexPDE software (version5.0.8) assuming an 8 μm radius spherical cell with the bottom of thecell flattened to a 5.5 μm adhesion spot where it contacts the glasssubstrate.

The above descriptions are those of the preferred embodiments of theinvention. Various modifications and variations are possible in light ofthe above teachings without departing from the spirit and broaderaspects of the invention. It is therefore to be understood that theclaimed invention may be practiced otherwise than as specificallydescribed. Any references to claim elements in the singular, forexample, using the articles “a,” “an,” “the,” or “said,” is not to beconstrued as limiting the element to the singular.

What is claimed as new and desired to be protected by Letters Patent ofthe United States is:
 1. A label-free method for the spatio-temporalmapping of secretions of antibodies from individual cells in real time,comprising: using a chip for localized surface plasmon resonance (LSPR)imaging, comprising a glass coverslip compatible for use in a standardmicroscope and at least one array of functionalized plasmonicnanostructures patterned onto the glass coverslip, wherein thefunctionalized plasmonic nanostructures comprise a binding reagent forthe secretions of antibodies; placing at least one cell on the chip; andspatially and temporally mapping secretions of antibodies from the cellusing LSPR imaging.
 2. The method of claim 1, additionally comprisingsimultaneously monitoring the cell secretions using transmitted lightimaging, fluorescence imaging, or any combination thereof.
 3. The methodof claim 1, wherein the functionalized plasmonic nanostructures comprisegold nanostructures.
 4. The method of claim 1, wherein the center of thecell of no more than 15 μm from an array.
 5. The method of claim 1,additionally comprising normalizing each array individually.
 6. Themethod of claim 1, additionally comprising using at least one array as acontrol array, wherein the control array is at least 65 μm away from thecell.